Project description:The present work comprises the study of wound pathogenic bacteria as part of a community. It considers the interactions of two different S. aureus isolates with B. thuringiensis and K. oxytoca; all of them isolated from the same chronic wound of a patient with epidermolysis bullosa. Particular focus has been given on the interactions of S. aureus with other microbes due to its high prevalence among chronic wounds. During cultivation, no species performed as dominant or inhibited the growth of one another. Mass spectrometry was used to explore the inherent relationships between the staphylococcal strains and the coexisting bacteria exproteomes. The analysis showed an important reduction in the amount of staphylococcal cytoplasmic proteins when co-cultured with K. oxytoca and B. thuringiensis, this decrement did not occur with klebsiella and bacillus proteins. Interestingly, K. oxytoca and B. thuringiensis seemed to have a more evident response towards the presence of S. aureus in the culture, while the opposite was not observed with the staphylococcal isolates. Genomic analysis revealed isolate t13595 hypermutable characteristics, placing the interactions between staphylococcal isolates in the context of a chronic wound. Overall, the nature of the exoproteome variations among cultures suggests that adaptive mechanisms differ in all strains.
Project description:Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites such as burned wounds, lung and urinary tract. To shed light on the expression rates of cytoplasmic P. aeruginosa proteins, commonly expressed by eleven different clinical isolates, absolute protein quantities were determined using P. aeruginosa PAO1 as a reference strain and employing a highly precise gel-free and data-independent LC-IMSE approach. Moreover, the metabolic diversity of these isolates has been investigated by 13C-metabolic flux analyses. 903 proteins were reproducibly identified and absolutely quantified for P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all of the tested clinical isolates. The vast majority of these proteins is expressed in constant amounts in all strains and exhibits a relatively low relative standard deviation. In contrast, the expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in isolates from burned wounds when compared to isolates from lung or urinary tract. Moreover, proteins involved in the uptake of iron and amino acids (i.e. HitA, BfrB, PA5217, BraC, PA5153) were found to be more abundant in urinary tract isolates compared to lung isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated analysis of proteome and fluxome did not indicate straightforward correlation between the amount of proteins and their flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments.
Project description:Background: S. aureus is one of the main pathogen involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable and ranges from subclinical to gangrenous mastitis. Such variability implies host as well as staphylococcal factors. This work is an in-depth characterization of S. aureus mastitis isolates to identify factors involved in mastitis severity. Methods and findings: We combined three “omic” approaches to comprehensively compare two clonally related S. aureus strains that were isolated from and shown to reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. The genomes of O11 and O46 were sequenced (Illumina technology) to determine their respective gene content and comparative transcriptomic and proteomic analyses were carried out on both strains grown in conditions mimicking mastitis context. High differences were highlighted in mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production. In particular, O11 overproduced exoproteins, including toxins and proteases when compared to O46. This was confirmed in 4 other S. aureus strains isolated from subclinical or clinical mastitis cases. Dose-dependant production of some staphylococcal factors seem to play a role in hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins or strain ability to deal with iron starvation constitute good targets for further research to better define the underlying mechanisms of mastitis severity. Conclusions: Differences observed in mastitis severity likely result from the ability of the strains to adapt and to express virulence factors in the mastitis context rather than from deep variations in gene content.
Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF.
Project description:Background: S. aureus is one of the main pathogen involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable and ranges from subclinical to gangrenous mastitis. Such variability implies host as well as staphylococcal factors. This work is an in-depth characterization of S. aureus mastitis isolates to identify factors involved in mastitis severity. Methods and findings: We combined three “omic” approaches to comprehensively compare two clonally related S. aureus strains that were isolated from and shown to reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. The genomes of O11 and O46 were sequenced (Illumina technology) to determine their respective gene content and comparative transcriptomic and proteomic analyses were carried out on both strains grown in conditions mimicking mastitis context. High differences were highlighted in mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production. In particular, O11 overproduced exoproteins, including toxins and proteases when compared to O46. This was confirmed in 4 other S. aureus strains isolated from subclinical or clinical mastitis cases. Dose-dependant production of some staphylococcal factors seem to play a role in hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins or strain ability to deal with iron starvation constitute good targets for further research to better define the underlying mechanisms of mastitis severity. Conclusions: Differences observed in mastitis severity likely result from the ability of the strains to adapt and to express virulence factors in the mastitis context rather than from deep variations in gene content. Expression of S. aureus O46 from subclinical mastitis and O11 from a lethal gangrenous mastitis were compared at two different times
Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to staphylococcal α-hemolysin oligomers (rAHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with 3nM of extracted oligomers (rAHL) for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.
Project description:Next-generation sequencing technologies have dramatically increased the rate at which new genomes are sequenced. Accordingly, automated-annotation programs have become adept at identifying and annotating protein coding regions, as well as common and conserved RNAs. Additionally, RNAseq techniques have advanced our ability to identify and annotate regulatory RNAs (sRNAs), which remain significantly understudied. Recently, our group catalogued and annotated all previously known and newly identified sRNAs in several Staphylococcus aureus strains. These complete annotation files now serve as tools to compare the sRNA content of S. aureus to other bacterial strains to investigate the conservation of their sRNomes. Accordingly, in this study we performed RNAseq on two staphylococcal species, S. epidermidis and S. carnosus, identifying 118 and 89 sRNAs in these organisms, respectively. The sRNA content of all three species were then compared to elucidate their common and species-specific sRNA content, identifying a core set between 53 and 36 sRNAs encoded in each organism. In addition, we determined that S. aureus has the largest set of unique sRNAs (137) while S. epidermidis has the fewest (25). Finally, we identify a highly conserved sequence and structural motif differentially represented within, yet common to, both S. aureus and S. epidermidis. Collectively, in this study, we uncover the sRNome common to three staphylococcal species, shedding light on sRNAs that are likely to be involved in basic physiological processes common to the genus. More significantly, we have identified species-specific sRNAs that are likely to influence the individual lifestyle and behavior of these diverse staphylococcal strains.